Versatile hydrophilic dyes

ABSTRACT

Dye-peptide conjugates useful for diagnostic imaging and therapy are disclosed. The dye-peptide conjugates include several cyanine dyes with a variety of bis- and tetrakis(carboxylic acid) homologues. The small size of the compounds allows more favorable delivery to tumor cells as compared to larger molecular weight imaging agents. The various dyes are useful over the range of 350-1300 nm, the exact range being dependent upon the particular dye. Use of dimethylsulfoxide helps to maintain the fluorescence of the compounds. The molecules of the invention are useful for diagnostic imaging and therapy, in endoscopic applications for the detection of tumors and other abnormalities and for localized therapy, for photoacoustic tumor imaging, detection and therapy, and for sonofluorescence tumor imaging, detection and therapy.

This application is a continuation of application Ser. No. 09/757,333filed Jan. 9, 2001 now U.S. Pat. No. 6,939,532, which is acontinuation-in-part of U.S. Pat. No. 6,183,726, Ser. No. 09/484,321,filed Jan. 18, 2000, each of which is expressly incorporated byreference herein its entirety.

FIELD OF THE INVENTION

This invention relates generally to compositions of cyanine dyebioconjugates with bioactive molecules for diagnosis and therapy,particularly, for visualization and detection of tumors.

BACKGROUND OF THE INVENTION

Several dyes that absorb and emit light in the visible and near-infraredregion of the electromagnetic spectrum are currently being used forvarious biomedical applications due to their biocompatibility, highmolar absorptivity, and/or high fluorescence quantum yields. The highsensitivity of the optical modality in conjunction with dyes as contrastagents parallels that of nuclear medicine, and permits visualization oforgans and tissues without the undesirable effect of ionizing radiation.

Cyanine dyes with intense absorption and emission in the near-infrared(NIR) region are particularly useful because biological tissues areoptically transparent in this region (B. C. Wilson, Optical propertiesof tissues. Encyclopedia of Human Biology, 1991, 5, 587-597). Forexample, indocyanine green, which absorbs and emits in the NIR region,has been used for monitoring cardiac output, hepatic functions, andliver blood flow (Y-L. He, et al., Measurement of blood volume usingindocyanine green measured with pulse-spectrometry: Its reproducibilityand reliability. Critical Care Medicine, 1998, 26(8), 1446-1451; J.Caesar, et al., The use of Indocyanine green in the measurement ofhepatic blood flow and as a test of hepatic function. Clin. Sci. 1961,21, 43-57), and its functionalized derivatives have been used toconjugate biomolecules for diagnostic purposes (R. B. Mujumdar, et al.,Cyanine dye labeling reagents: Sulfoindocyanine succinimidyl esters.Bioconjugate Chemistry, 1993, 4(2), 105-111; U.S. Pat. No. 5,453,505; WO98/48846; WO 98/22146; WO 96/17628; WO 98/48838).

A major drawback in the use of cyanine dye derivatives is the potentialfor hepatobiliary toxicity resulting from the rapid clearance of thesedyes by the liver (G. R. Cherrick, et al., Indocyanine green:Observations on its physical properties, plasma decay, and hepaticextraction. J. Clinical Investigation, 1960, 39, 592-600). This isassociated with the tendency of cyanine dyes in solution to formaggregates, which could be taken up by Kupffer cells in the liver.

Various attempts to obviate this problem have not been very successful.Typically, hydrophilic peptides, polyethyleneglycol or oligosaccharideconjugates have been used, but these resulted in long-circulatingproducts, which are eventually still cleared by the liver. Another majordifficulty with current cyanine and indocyanine dye systems is that theyoffer a limited scope in the ability to induce large changes in theabsorption and emission properties of these dyes. Attempts have beenmade to incorporate various heteroatoms and cyclic moieties into thepolyene chain of these dyes (L. Strekowski, et al., Substitutionreactions of a nucleofugal group in hetamethine cyanine dyes. J. Org.Chem., 1992, 57, 4578-4580; N. Narayanan and G. Patonay, A new methodfor the synthesis of heptamethine cyanine dyes: Synthesis of new nearinfrared fluorescent labels. J. Org. Chem., 1995, 60, 2391-2395; U.S.Pat. Nos. 5,732,104; 5,672,333; and 5,709,845), but the resulting dyesystems do not show large differences in absorption and emission maxima,especially beyond 830 nm where photoacoustic diagnostic applications arevery sensitive. They also possess a prominent hydrophobic core, whichenhances liver uptake. Further, most cyanine dyes do not have thecapacity to form starburst dendrimers, which are useful in biomedicalapplications.

For the purpose of tumor detection, many conventional dyes are usefulfor in vitro applications because of their highly toxic effect on bothnormal and abnormal tissues. Other dyes lack specificity for particularorgans or tissues and, hence, must be attached to bioactive carrierssuch as proteins, peptides, carbohydrates, and the like to deliver thedyes to specific regions in the body. Several studies on the use of nearinfrared dyes and dye-biomolecule conjugates have been published (G.Patonay and M. D. Antoine, Near-Infrared Fluorogenic Labels: NewApproach to an Old Problem, Analytical Chemistry, 1991, 63:321A-327A andreferences therein; M. Brinkley, A Brief Survey of Methods for PreparingProtein Conjugates with Dyes, Haptens, and Cross-Linking Reagents,Perspectives in Bioconjugate Chemistry, 1993, pp. 59-70, C. Meares (Ed),ACS Publication, Washington, D.C.; J. Slavik, Fluorescent Probes inCellular and Molecular Biology, 1994, CRC Press, Inc.; U.S. Pat. No.5,453,505; WO 98/48846; WO 98/22146; WO 96/17628; WO 98/48838).

Of particular interest is the targeting of tumor cells with antibodiesor other large protein carriers such as transferrin as delivery vehicles(A. Becker et al., “Transferrin Mediated Tumor Delivery of ContrastMedia for Optical Imaging and Magnetic Resonance Imaging”, BiomedicalOptics meeting, Jan. 23-29, 1999, San Jose, Calif.). Such an approachhas been widely used in nuclear medicine applications. Its majoradvantage is the retention of a carrier's tissue specificity, since themolecular volume of the dye is substantially smaller than the carrier.However, this approach does have some serious limitations in that thediffusion of high molecular weight bioconjugates to tumor cells ishighly unfavorable, and is further complicated by the net positivepressure in solid tumors (R. K. Jain, Barriers to Drug Delivery in SolidTumors, Scientific American, 1994, 271:58-65. Furthermore, many dyes ingeneral, and cyanine dyes in particular, tend to form aggregates inaqueous media that lead to fluorescence quenching.

Therefore, there is a need for dyes that could prevent dye aggregationin solution, that are predisposed to form dendrimers, that are capableof absorbing or emitting beyond 800 nm, that possess desirablephotophysical properties, and that are endowed with tissue-specifictargeting capability.

SUMMARY OF THE INVENTION

The invention is directed to compositions, and methods of preparing thecompositions, of low molecular weight biomolecule-dye conjugates toenhance tumor detection. The inventive compositions preserve thefluorescence efficiency of the dye molecules, do not aggregate insolution, form starburst dendrimers, are capable of absorbing oremitting light in the near infrared region (beyond 800 nm), and can berendered tissue-specific.

In one embodiment, the inventive composition comprises cyanine dyes ofgeneral formula 1

wherein W³ and X³ may be the same or different and are selected from thegroup consisting of —CR¹R², —O—, —NR³, —S—, and —Se; Y³ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,—(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z³ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₁ is a single or adouble bond; B₁, C₁, and D₁ may the same or different and are selectedfrom the group consisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl,NR³, and —C═O; A₁, B₁, C₁, and D₁ may together form a 6- to 12-memberedcarbocyclic ring or a 6- to 12-membered heterocyclic ring optionallycontaining one or more oxygen, nitrogen, or sulfur atom; a₃ and b₃ areindependently from 0 to 5; R¹ to R⁴, and R²⁹ to R³⁷ are independentlyselected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl,C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀ aminoalkyl, cyano, nitro, halogen,saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H,—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide, a protein, a cell, an antibody,an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, adrug, a drug mimic, a hormone, a metal chelating agent, a radioactive ornonradioactive metal complex, and an echogenic agent; a and c areindependently from 1 to 20; and b and d are independently from 1 to 100.

In a second embodiment, the inventive composition comprises cyanine dyesof general formula 2

wherein W⁴ and X⁴ may be the same or different and are selected from thegroup consisting of —CR¹R², —O—, —NR³, —S—, and —Se; Y⁴ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁴ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₂ is a single or adouble bond; B₂, C₂, and D₂ may the same or different and are selectedfrom the group consisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl,NR³, and —C═O; A₂, B₂, C₂, and D₂ may together form a 6- to 12-memberedcarbocyclic ring or a 6- to 12-membered heterocyclic ring optionallycontaining one or more oxygen, nitrogen, or sulfur atom; a₄ and b₄independently vary from 0 to 5; R¹ to R⁴, and R⁴⁵ to R⁵⁷ areindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₅-C₂₀ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyalkoxyalkyl, C₁-C₂₀polyhydroxyalkyl, C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀ aminoalkyl, cyano,nitro, halogen, saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—OH,—(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—NHCO—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide, a protein, a cell, an antibody,an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, adrug, a drug mimic, a hormone, a metal chelating agent, a radioactive ornonradioactive metal complex, and an echogenic agent; a and c areindependently from 1 to 20; and b and d are independently from 1 to 100.

In a third embodiment, the inventive composition comprises cyanine dyesof general formula 3

wherein W⁵ and X⁵ may be the same or different and are selected from thegroup consisting of —CR¹R², —O—, —NR³, —S—, and —Se; Y⁵ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or adouble bond; B₃, C₃, and D₃ may the same or different and are selectedfrom the group consisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl,NR³, and —C═O; A₃, B₃, C₃, and D₃ may together form a 6- to 12-memberedcarbocyclic ring or a 6- to 12-membered heterocyclic ring optionallycontaining one or more oxygen, nitrogen, or sulfur atom; a₅ isindependently from 0 to 5; R¹ to R⁴, and R⁵⁸ to R⁶⁶ are independentlyselected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl,C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀ aminoalkyl, cyano, nitro, halogen,saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H,—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide, a protein, a cell, an antibody,an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, adrug, a drug mimic, a hormone, a metal chelating agent, a radioactive ornonradioactive metal complex, and an echogenic agent; a and c areindependently from 1 to 20; and b and d are independently from 1 to 100.

In a fourth embodiment, inventive composition comprises cyanine dyes ofgeneral formula 4

wherein W⁶ and X⁶ may be the same or different and are selected from thegroup consisting of —CR¹R², —O—, —NR³, —S—, and —Se; Y⁶ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁶ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₄ is a single or adouble bond; B₄, C₄, and D₄ may the same or different and are selectedfrom the group consisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl,NR³, and —C═O; A₄, B₄, C₄, and D₄ may together form a 6- to 12-memberedcarbocyclic ring or a 6- to 12-membered heterocyclic ring optionallycontaining one or more oxygen, nitrogen, or sulfur atom; a₆ isindependently from 0 to 5; R¹ to R⁴, and R⁶⁷ to R⁷⁹ are independentlyselected from the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl,C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀ aminoalkyl, cyano, nitro, halogen,saccharide, peptide, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H,—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH or—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide, a protein, a cell, an antibody,an antibody fragment, a saccharide, a glycopeptide, a peptidomimetic, adrug, a drug mimic, a hormone, a metal chelating agent, a radioactiveand nonradioactive metal complex, and an echogenic agent; a and c areindependently from 1 to 20; and b and d are independently from 1 to 100.

The invention will be further appreciated in light of the followingfigures, detailed description, and examples.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 shows the reaction pathway for the synthesis of bis-carboxylicacid cyanine dyes.

FIG. 2 shows the reaction pathway for the synthesis of tetracarboxylicacid cyanine dyes.

FIG. 3 shows the reaction pathway for the synthesis ofpolyhydroxycarboxylic acid dyes.

FIG. 4 shows the reaction pathway for the synthesis of non-aggregatingcyanine dyes.

FIG. 5 shows the reaction pathway for the synthesis of long wavelengthabsorbing dyes.

FIG. 6 shows the reaction pathway for the synthesis of cyanine dyebioconjugates.

FIGS. 7A-F represent images at 2 minutes and 30 minutes post injectionof indocyanine green (ICG) into rats with various tumors.

FIGS. 8A-B show a comparison of the uptake of ICG (FIG. 8A) and Cytate 1(FIG. 8B) in rats with the pancreatic acinar carcinoma (CA20948).

FIGS. 9A-B show images of rats with the pancreatic acinar carcinoma(CA20948) 45 minutes (FIG. 9A) and 27 hours (FIG. 9B) post injection ofCytate 1.

FIG. 10 is an image of individual organs taken from a rat withpancreatic acinar carcinoma (CA20948) about 24 hours after injectionwith Cytate 1.

FIG. 11 is an image of bombesinate in an AR42-J tumor-bearing rat 22hours after injection.

FIG. 12 is the clearance profile of Cytate 1 from the blood of a normalrat.

FIG. 13 is the clearance profile of Cytate 1 from the blood of apancreatic tumor-bearing rat.

FIG. 14 is the clearance profile of Cytate 2 from the blood of a normalrat.

FIG. 15 is the clearance profile of Cytate 2 from the blood of apancreatic tumor-bearing rat.

FIG. 16 is the clearance profile of Cytate 4 from the blood of a normalrat.

DETAILED DESCRIPTION OF THE INVENTION

The novel compositions of the present invention comprising dyes offormulas 1 to 4 offer significant advantages over those currentlydescribed in the art. These inventive dyes form starburst dendrimerswhich prevent aggregation in solution by preventing intramolecular andintermolecular ordered hydrophobic interactions, and have multipleattachment sites proximal to the dye chromophore for ease of formingbioactive molecules. The presence of rigid and extended chromophorebackbone enhances their fluorescence quantum yield and extends theirmaximum absorption beyond 800 nm. Conjugation of biomolecules to thesedyes is readily achievable.

The inventive bioconjugates of the present invention also exploit thesymmetric nature of the cyanine and indocyanine dye structures byincorporating one to ten receptor targeting groups in close proximity toeach other, such that the receptor binding can be greatly enhanced dueto a cooperative effect. Accordingly, several cyanine dyes containingone or more targeting domains have been prepared and tested in vivo forbiological activity.

The inventive dye-bioconjugates of formulas 1 to 4 are useful forvarious biomedical applications. These include, but are not limited to,tomographic imaging of organs, monitoring of organ functions, coronaryangiography, fluorescence endoscopy, detection, imaging, and therapy oftumors, laser assisted guided surgery, photoacoustic methods, andsonofluorescent methods.

Specific embodiments to accomplish some of the aforementioned biomedicalapplications are given below. The novel dyes of the present inventionare prepared according the methods well known in the art and areillustrated in FIGS. 1-5.

FIG. 1 illustrates the synthetic scheme for bis-carboxylic acid cyaninedyes, where A=CH₂ or CH₂OCH₂; R═COOH; R′=COOH, NHFmoc; CO₂t-Bu; SO₃ ⁻;R₁=R₂=H (Formula 1) or R₁, R₂=fused phenyl (Formula 2).

FIG. 2 illustrates the synthetic scheme for tetracarboxylic acid cyaninedyes, where A=CH₂ or CH₂OCH₂; R₁=R₂=H (Formula 1) or R₁, R₂=fused phenyl(Formula 2).

FIG. 3 illustrates the synthetic scheme for polyhydroxy-carboxylic acidcyanine dyes.

FIG. 4 illustrates the synthetic scheme for non-aggregating cyaninedyes.

FIG. 5 illustrates the synthetic scheme for long wavelength-absorbing“tu nable” cyanine dyes.

In one embodiment, the inventive bioconjugates have the Formula 1,wherein W³ and X³ may be the same or different and are selected from thegroup consisting of —C(CH₃)₂, —C((CH₂)_(a)OH)CH₃, —C((CH₂)_(a)OH)₂,—C((CH₂)_(a)CO₂H)CH₃, —C((CH₂)_(a)CO₂H)₂, —C((CH₂)_(a)NH₂)CH₃,—C((CH₂)_(a)NH₂)₂, —C((CH₂)_(a)NR³R⁴)₂, —NR³, and —S—; Y³ is selectedfrom the group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z³ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₁ is a single or a double bond; B₁, C₁, andD₁ are independently selected from the group consisting of —O—, —S—,NR³, (CH₂)_(a)—CR¹R², and —CR¹; A₁, B₁, C₁, and D₁ may together form a6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclicring optionally containing one or more oxygen, nitrogen, or sulfur atom;a₃ and b₃ are independently from 0 to 3; R¹ to R⁴, and R²⁹ to R³⁷ areindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₅-C₁₂ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂polyhydroxyaryl, C₁-C₁₀ aminoalkyl, mono- or oligosaccharide, peptidewith 2 to 30 amino acid units, —CH₂(CH₂OCH₂)_(b)—CH₂—OH,—(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—NHCO—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide containing 2 to 30 amino acidunits, an antibody, a mono- or oligosaccharide, a glycopeptide, a metalchelating agent, a radioactive or nonradioactive metal complex, and anechogenic agent; a and c are independently from 1 to 10; and b and d areindependently from 1 to 30.

In a second embodiment, the inventive bioconjugates have the generalFormula 2, wherein W⁴ and X⁴ may be the same or different and areselected from the group consisting of —C(CH₃)₂, —C((CH₂)_(a)OH)CH₃,—C((CH₂)_(a)OH)₂, —C((CH₂)_(a)CO₂H)CH₃, —C((CH₂)_(a)CO₂H)₂,—C((CH₂)_(a)NH₂)CH₃, C((CH₂)_(a)NH₂)₂, —C((CH₂)_(a)NR³R⁴)₂, —NR³, and—S—; Y⁴ is selected from the group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁴ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₂ is a single or a double bond; B₂, C₂, andD₂ are independently selected from the group consisting of —O—, —S—,NR³, (CH₂)_(a)—CR¹R², and —CR¹; A₂, B₂, C₂, and D₂ may together form a6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclicring optionally containing one or more oxygen, nitrogen, or sulfur atom;a₄ and b₄ are independently from 0 to 3; R¹ to R⁴, and R⁴⁵ to R⁵⁷ areindependently selected from the group consisting of hydrogen, C₁-C₁₀alkyl, C₅-C₁₂ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂polyhydroxyaryl, C₁-C₁₀ aminoalkyl, mono- or oligosaccharide, peptidewith 2 to 30 amino acid units, —CH₂(CH₂OCH₂)_(b)—CH₂—OH,—(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—NHCO—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide containing 2 to 30 amino acidunits, an antibody, a mono- or oligosaccharide, a glycopeptide, a metalchelating agent, a radioactive or nonradioactive metal complex, and anechogenic agent; a and c are independently from 1 to 10; and b and d areindependently from 1 to 30.

In a third embodiment, the inventive bioconjugates have the generalFormula 3, wherein W⁵ and X⁵ may be the same or different and areselected from the group consisting of —C(CH₃)₂, —C((CH₂)_(a)OH)CH₃,—C((CH₂)_(a)OH)₂, —C((CH₂)_(a)CO₂H)CH₃, —C((CH₂)_(a)CO₂H)₂,—C((CH₂)_(a)NH₂)CH₃, —C((CH₂)_(a)NH₂)₂, —C((CH₂)_(a)NR³R⁴)₂, —NR³, and—S—; Y⁵ is selected from the group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or a double bond; B₃, C₃, andD₃ are independently selected from the group consisting of —O—, —S—,NR³, (CH2)_(a)—CR¹R², and —CR¹; A₃, B₃, C₃, and D₃ may together form a6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclicring optionally containing one or more oxygen, nitrogen, or sulfur atom;a₅ is from 0 to 3; R¹ to R⁴, and R⁵⁸ to R⁶⁶ are independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acidunits, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide containing 2 to 30 amino acidunits, an antibody, a mono- or oligosaccharide, a glycopeptide, a metalchelating agent, a radioactive or nonradioactive metal complex, and anechogenic agent; a and c are independently from 1 to 10; and b and d areindependently from 1 to 30.

In a fourth embodiment, the inventive bioconjugates have the generalFormula 4, wherein W⁶ and X⁶ may be the same or different and areselected from the group consisting of —C(CH₃)₂, —C((CH₂)_(a)OH)CH₃,—C((CH₂)_(a)OH)₂, —C((CH₂)_(a)CO₂H)CH₃, C((CH₂)_(a)CO₂H)₂,—C((CH₂)_(a)NH₂)CH₃, —C((CH₂)_(a)NH₂)₂, C((CH₂)_(a)NR³R⁴)₂, —NR³, and—S—; Y⁶ is selected from the group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁶ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₄ is a single or a double bond; B₄, C₄, andD₄ are independently selected from the group consisting of —O—, —S—,NR³, (CH₂)_(a)—CR¹R², and —CR¹; A₄, B₄, C₄, and D₄ may together form a6- to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclicring optionally containing one or more oxygen, nitrogen, or sulfur atom;a₆ is from 0 to 3; R¹ to R₄, and R⁶⁷ to R⁷⁹ are independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acidunits, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of a bioactive peptide containing 2 to 30 amino acidunits, an antibody, a mono- or oligosaccharide, a glycopeptide, a metalchelating agent, a radioactive or nonradioactive metal complex, and anechogenic agent; a and c are independently from 1 to 10; and b and d areindependently from 1 to 30.

This invention is also related to the method of conjugating theinventive dyes to peptides or biomolecules by solid phase or solutionsynthesis methods. FIG. 6 illustrates the synthetic scheme forbioconjugates incorporating the cyanine dyes of FIGS. 1-5, usingautomated peptide synthesis in a solid support, is shown in FIG. 6,where A=CH₂ or CH₂OCH₂; R₁=R₂=H (Formula 1) or R₁, R₂=fused phenyl(Formula 2); AA=amino acids; R=CONH peptide; R′=R (bis conjugate) orCOOH (mono conjugate); P=solid support; P′=presence or absence dependson R′ definition.

This invention is also related to the method of preventing fluorescencequenching. It is known that cyanine dyes generally form aggregates inaqueous media, leading to fluorescence quenching. Where the presence ofa hydrophobic core in the dyes leads to fluorescence quenching, theaddition of a biocompatible organic solvent, such as 1-50%dimethylsulfoxide (DMSO) for example, restored fluorescence bypreventing aggregation and allowed in vivo organ visualization.

The inventive dye-biomolecule conjugates are used for opticaltomographic, endoscopic, photoacoustic and sonofluorescent applicationsfor the detection and treatment of tumors and other abnormalities.

Dye-biomolecule conjugates are also used for localized therapy. This maybe accomplished by attaching a porphyrin or other photodynamic therapyagent to a bioconjugate, shining light of an appropriate wavelength toactivate the agent, and detecting and/or treating the abnormality.

The inventive conjugates can also be used for the detection of thepresence of tumors and other abnormalities by monitoring the bloodclearance profile of the conjugates, for laser assisted guided surgeryfor the detection of small micrometastases of, e.g., somatostatinsubtype 2 (SST-2) positive tumors, upon laparoscopy, and for diagnosisof atherosclerotic plaques and blood clots.

The compositions of the invention can be formulated into diagnostic andtherapeutic compositions for enteral or parenteral administration. Thesecompositions contain an effective amount of the dye along withconventional pharmaceutical carriers and excipients appropriate for thetype of administration contemplated. For example, parenteralformulations advantageously contain the inventive agent in a sterileaqueous solution or suspension. Parenteral compositions may be injecteddirectly or mixed with a large volume parenteral composition forsystemic administration. Such solutions also may containpharmaceutically acceptable buffers and, optionally, electrolytes suchas sodium chloride.

Formulations for enteral administration may vary widely, as is wellknown in the art. In general, such formulations are liquids, whichinclude an effective amount of the inventive agent in aqueous solutionor suspension. Such enteral compositions may optionally include buffers,surfactants, thixotropic agents, and the like. Compositions for oraladministration may also contain flavoring agents and other ingredientsfor enhancing their organoleptic qualities.

The diagnostic compositions are administered in doses effective toachieve the desired enhancement. Such doses may vary widely, dependingupon the particular dye employed, the organs or tissues to be imaged,the imaging equipment being used, and the like. The diagnosticcompositions of the invention are used in the conventional manner. Thecompositions may be administered to a patient, typically a warm-bloodedanimal, either systemically or locally to the organ or tissue to beimaged, and the patient is then subjected to the imaging procedure.

The inventive compositions and methods represent an important approachto the synthesis and use of novel cyanine and indocyanine dyes with avariety of photophysical and chemical properties. The combination alsorepresents an important approach to the use of small molecular targetinggroups to image tumors by optical methods. The invention is furtherdetailed in the following Examples, which are offered by way ofillustration and are not intended to limit the scope of the invention inany manner.

EXAMPLE 1 Synthesis of Bis(ethylcarboxymethyl)indocyanine Dye (FIG. 1,R₁, R₂=Fused Phenyl; A=CH₂, n=1 and R=R′=CO₂H)

A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (9.1 g, 43.58 mmoles)and 3-bromopropanoic acid (10.0 g, 65.37 mmoles) in 1,2-dichlorobenzene(40 mL) was heated at 110° C. for 12 hours. The solution was cooled toroom temperature and the red residue obtained was filtered and washedwith acetonitrile:diethyl ether (1:1) mixture. The solid obtained wasdried under vacuum to give 10 g (64%) of light brown powder. A portionof this solid (6.0 g; 16.56 mmoles), glutaconaldehyde dianilmonohydrochloride (2.36 g, 8.28 mmoles), and sodium acetate trihydrate(2.93 g, 21.53 mmoles) in ethanol (150 mL) were refluxed for 90 minutes.After evaporating the solvent, 40 mL of 2 N aqueous HCl was added to theresidue. The mixture was centrifuged and the supernatant was decanted.This procedure was repeated until the supernatant became nearlycolorless. About 5 mL of water:acetonitrile (3:2) mixture was added tothe solid residue and lyophilized to obtain 2 g of dark green flakes.The purity of the compound was established with ¹H-NMR and liquidchromatography-mass spectroscopy (LC-MS).

EXAMPLE 2 Synthesis of Bis(pentylcarboxymethyl)indocyanine Dye (FIG. 1,R₁, R₂=Fused Phenyl; A=CH₂, n=4 and R=R′=CO₂H)

A mixture of 1,1,2-trimethyl-[1H]-benz[e]indole (20 g, 95.6 mmoles) and6-bromohexanoic acid (28.1 g, 144.1 mmoles) in 1,2-dichlorobenzene (250mL) was heated at 110° C. for 12 hours. The green solution was cooled toroom temperature and the brown solid precipitate formed was collected byfiltration. After washing the solid with 1,2-dichlorobenzene and diethylether, the brown powder obtained (24 g, 64%) was dried under vacuum atroom temperature. A portion of this solid (4.0 g; 9.8 mmoles),glutaconaldehyde dianil monohydrochloride (1.4 g, 5 mmoles) and sodiumacetate trihydrate (1.8 g, 12.9 mmoles) in ethanol (80 mL) were refluxedfor 1 hour. After evaporating the solvent, 20 mL of 2 N aqueous HCl wasadded to the residue. The mixture was centrifuged and the supernatantwas decanted. This procedure was repeated until the supernatant becamenearly colorless. About 5 mL of water:acetonitrile (3:2) mixture wasadded to the solid residue and lyophilized to obtain about 2 g of darkgreen flakes. The purity of the compound was established with ¹H-NMR andLC-MS.

EXAMPLE 3 Synthesis of Bisethylcarboxymethylindocyanine Dye (FIG. 1,R₁=R₂=H; A=CH₂, n=1 and R=R′=CO₂H)

This compound was prepared as described in Example 1 except that1,1,2-trimethylindole was used as the starting material.

EXAMPLE 4 Synthesis of Bis(hexaethyleneglycolcarboxymethyl)indocyanineDye (FIG. 1, R₁=R₂=fused phenyl; A=CH₂OCH₂, n=6 and R=R′=CO₂H)

This compound was prepared as described in Example 1 except thatω-bromohexaoxyethyleneglycolpropiolic acid was used in place ofbromopropanoic acid and the reaction was carried out in1,2-dimethoxypropane.

EXAMPLE 5 Synthesis of Bisethylcarboxymethylindocyanine Dye (FIG. 2,R₁=R₂=Fused Phenyl; A=CH₂, and n=0)

A solution of 50 ml of dimethylformamide and benzyl bromoacetate (16.0g, 70 mmol) was stirred in a 100 ml three-neck flask. Solid potassiumbicarbonate (7.8 g, 78 mmol) was added. The flask was purged with argonand cooled to 0° C. with an ice bath. To the stirring mixture was addeddropwise a solution of ethanolamine (1.9 g, 31 mmol) and 4 ml ofdimethylformamide over 5 minutes. After the addition was complete themixture was stirred for 1 hour at 0° C. The ice bath was removed and themixture was stirred at room temperature overnight. The reaction mixturewas partitioned between 100 ml of methylene chloride and 100 ml ofsaturated sodium bicarbonate solution. The layers were separated and themethylene chloride layer was again washed with 100 ml of saturatedsodium bicarbonate solution. The combined aqueous layers were extractedtwice with 25 ml of methylene chloride. The combined methylene chloridelayers were washed with 100 ml of brine, and dried over magnesiumsulfate. The methylene chloride was removed with aspirator vacuum atabout 35° C., and the remaining dimethylformamide was removed withvacuum at about 45° C. The crude material was left on a vacuum lineovernight at room temperature.

The crude material was then dissolved in 100 ml of methylene chloride atroom temperature. Triphenylphosphine (8.91 g, 34 mmol) was added anddissolved with stirring. An argon purge was started and the mixture wascooled to 0° C. with an ice bath. The N-bromosuccinimide (6.05 g, 34mmol) was added portionwise over two minutes. The mixture was stirredfor 1.5 hours at 0° C. The methylene chloride was removed with vacuumand gave a purple oil. This oil was triturated with 200 ml of ether withconstant manual stirring. During this time the oil became very thick.The ether solution was decanted and the oil was triturated with 100 mlof ether. The ether solution was decanted and the oil was againtriturated with a 100 ml portion of ether. The ether was decanted andthe combined ether solution was allowed to stand for about two hours toallow the triphenylphosphine oxide to crystallize. The ether solutionwas decanted from the crystals and the solid was washed with 100 ml ofether. The volume of the combined ether extracts was reduced with vacuumuntil a volume of about 25 ml was obtained. This was allowed to standovernight at 0° C. Ether (10 ml) was added to the cold mixture, whichwas mixed to suspend the solid. The mixture was percolated through acolumn of 45 g of silica gel and eluted with ether, and 75 ml fractionswere collected. The fractions that contained product, as determined bythin layer chromatography, were pooled and the ether was removed withvacuum. This yielded 10.1 g of crude product. The material was flashchromatographed on silica gel wi with hexane, changing to 9:1hexane:ether. The product-containing fractions were pooled and thesolvents removed with vacuum. This yielded 7.4 g (57% yield) of pureproduct.

A mixture of 10% palladium on carbon (1 g) and a solution of the benzylester (10 g) in 150 ml of methanol was hydrogenolyzed at 25 psi for twohours. The mixture was filtered over celite and the residue was washedwith methanol. The solvent was evaporated to give a viscous oil inquantitative yield.

Reaction of the bromide with 1,1,2-trimethyl-[1H]-benz[e]indole wascarried out as described in Example 1.

EXAMPLE 6 Bis(ethylcarboxymethyldihydroxyl)indocyanine Dye (FIG. 3)

The hydroxy-indole compound is readily prepared by a known method (P. L.Southwick et al., One pot Fischer synthesis of(2,3,3-trimethyl-3-H-indol-5-yl)-acetic acid derivatives asintermediates for fluorescent biolabels. Org. Prep. Proced. Int. Briefs,1988, 20(3), 279-284). Reaction of p-carboxymethylphenylhydrazinehydrochloride (30 mmol, 1 equiv.) and 1,1-bis(hydroxymethyl)propanone(45 mmole, 1.5 equiv.) in acetic acid (50 mL) at room temperature for 30minutes and at reflux for one minute gives(3,3-dihydroxymethyl-2-methyl-3-H-indol-5-yl)-acetic acid as a solidresidue. The reaction of 3-bromopropyl-N,N-bis(carboxymethyl)amine,which was prepared as described in Example 5, with the intermediateindole and subsequent reaction of the indole intermediate withglutaconaldehyde dianil monohydrochloride (see Example 1) gives thedesired product.

EXAMPLE 7 Synthesis of Bis(propylcarboxymethyl)indocyanine Dye (FIG. 4)

The intermediate 2-chloro-1-formyl-3-hydroxymethylenecyclohexane wasprepared as described in the literature (G. A. Reynolds and K. H.Drexhage, Stable heptamethine pyrylium dyes that absorb in the infrared.J. Org. Chem., 1977, 42(5), 885-888). Equal volumes (40 mL each) ofdimethylformamide (DMF) and dichloromethane were mixed and the solutionwas cooled to −10° C. in an acetone-dry ice bath. Under argonatmosphere, phosphorus oxychloride (40 mL) in dichloromethane was addeddropwise to the cool DMF solution, followed by the addition of 10 g ofcyclohexanone. The resulting solution was allowed to warm to roomtemperature and was refluxed for six hours. After cooling to roomtemperature, the mixture was poured into ice-cold water and stored at 4°C. for twelve hours. About 8 g of yellow powder was obtained afterfiltration. Condensation of the cyclic dialdehyde with the indoleintermediate is carried out as described in Example 1. Furtherfunctionalization of the dye with bis isopropylidene acetal protectedmonosaccharide was accomplished by the method described in theliterature (J. H. Flanagan, et al., Near infrared heavy-atom-modifiedfluorescent dyes for base-calling in DNA-sequencing application usingtemporal discrimination. Anal. Chem., 1998, 70(13), 2676-2684).

EXAMPLE 8 Synthesis of Bis(ethylcarboxymethyl)indocyanine Dye (FIG. 5)

These dyes are prepared as described in Example 7. These dyes absorb inthe infrared region. The typical example shown in FIG. 5 has anestimated absorption maximum at 1036 nm.

EXAMPLE 9 Synthesis of Peptides

The procedure described below is for the synthesis of Octreotate. Theamino acid sequence of Octreotate is:D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr (SEQ ID NO:1), wherein Cys′indicates the presence of an intramolecular disulfide bond between twocysteine amino acids. Other peptides of this invention were prepared bya similar procedure with slight modifications in some cases.

The octapeptide was prepared by an automated fluorenylmethoxycarbonyl(Fmoc) solid phase peptide synthesis using a commercial peptidesynthesizer from Applied Biosystems (Model 432A SYNERGY PeptideSynthesizer). The first peptide cartridge contained Wang resinpre-loaded with Fmoc-Thr on 25 μmole scale. Subsequent cartridgescontained Fmoc-protected amino acids with side chain protecting groupsfor the following amino acids: Cys(Acm), Thr(t-Bu), Lys(Boc), Trp(Boc)and Tyr(t-Bu). The amino acid cartridges were placed on the peptidesynthesizer and the product was synthesized from the C- to theN-terminal position. The coupling reaction was carried out with 75μmoles of the protected amino acids in the presence of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU)/N-hydroxybenzotriazole (HOBt). The Fmoc protecting group wasremoved with 20% piperidine in dimethylformamide. After the synthesiswas complete, the thiol group was cyclized with thalliumtrifluoroacetate and the product was cleaved from the solid support witha cleavage mixture containing trifluoroacetic acid (85%):water(5%):phenol (5%):thioanisole (5%) for six hours. The peptide wasprecipitated with t-butyl methyl ether and lyophilized withwater:acetonitrile (2:3) mixture. The peptide was purified by HPLC andanalyzed with LC/MS.

Octreotide, D-Phe-Cys′-Tyr-D-Trp-Lys-Thr-Cys′-Thr-OH (SEQ ID NO:2),wherein Cys′ indicates the presence of an intramolecular disulfide bondbetween two cysteine amino acids, was prepared by the same procedure.

Bombesin analogs were prepared by the same procedure except thatcyclization with thallium trifluoroacetate was not needed. Side-chaindeprotection and cleavage from the resin was carried out with 50 μL eachof ethanedithiol, thioanisole and water, and 850 μL of trifluoroaceticacid. Two analogues were prepared:Gly-Ser-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (SEQ ID NO:3) andGly-Asp-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂ (SEQ ID NO:4).

Cholecystokinin octapeptide analogs were prepared as described forOctreotate without the cyclization step. Three analogs were prepared:Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH₂ (SEQ ID NO:5);Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂ (SEQ ID NO:6); andD-Asp-Tyr-Nle-Gly-Trp-Nle-Asp-Phe-NH₂ (SEQ ID NO:7), where Nle isnorleucine.

A neurotensin analog, D-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu (SEQ ID NO:8),was prepared as described for Octreotate without the cyclization step.

EXAMPLE 10 Synthesis of Peptide-Dye Conjugates (FIG. 6)

The method described below is for the synthesis of Octreotate-cyaninedye conjugates, but a similar procedure is used for the synthesis ofother peptide-dye conjugates.

Octreotate was prepared as described in Example 9 but the peptide wasnot cleaved from the solid support and the N-terminal Fmoc group of Phewas retained. The thiol group was cyclized with thalliumtrifluoroacetate and the Phe was deprotected to liberate the free amine.Bisethylcarboxymethylindocyanine dye (53 mg, 75 μmoles) was added to anactivation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO(375 μL), and 0.2 M solution of diisopropylethylamine in DMSO (375 μL).The activation was complete in about 30 minutes and the resin-boundpeptide (25 μmoles) was added to the dye. The coupling reaction wascarried out at room temperature for three hours. The mixture wasfiltered and the solid residue was washed with DMF, acetonitrile andTHF. After drying the green residue, the peptide was cleaved from theresin and the side chain protecting groups were removed with a mixtureof 85% trifluoroacetic acid, 2.5% water, 2.5% thioanisole and 2.5%phenol. The resin was filtered and cold t-butyl methyl ether (MTBE) wasused to precipitate the dye-peptide conjugate, which was dissolved in anacetonitrile:water (2:3) mixture and lyophilized. The product waspurified by HPLC to give themonoOctreotate-Bisethylcarboxymethylindocyanine dye (Cytate 1, 80%) andthe bis Octreotate-Bisethylcarboxymethylindocyanine dye (Cytate 2, 20%).The monoOctreotate conjugate is obtained almost exclusively (>95%) overthe bis conjugate by reducing the reaction time to two hours. However,this also leads to incomplete reaction, and the free Octreotate must becarefully separated from the dye conjugate in order to avoid saturationof the receptors by the non-dye conjugated peptide.

Octreotate-bispentylcarboxymethylindocyanine dye was prepared asdescribed above with some modifications.Bispentylcarboxymethylindocyanine dye (60 mg, 75 μmoles) was added to anactivation reagent consisting of a 0.2 M solution of HBTU/HOBt in DMSO(400 μL), and a 0.2 M solution of diisopropylethylamine in DMSO (400μL). The activation was complete in about 30 minutes and the resin-boundpeptide (25 μmoles) was added to the dye. The reaction was carried outat room temperature for three hours. The mixture was filtered and thesolid residue was washed with DMF, acetonitrile and THF. After dryingthe green residue, the peptide was cleaved from the resin and the sidechain protecting groups were removed with a mixture of 85%trifluoroacetic acid, 2.5% water, 2.5% thioanisole and 2.5% phenol. Theresin was filtered and cold t-butyl methyl ether (MTBE) was used toprecipitate the dye-peptide conjugate, which was dissolved in anacetonitrile:water (2:3) mixture and lyophilized. The product waspurified by HPLC to give Octreotate-1,1,2-trimethyl-[1H]-benz[e]indolepropanoic acid conjugate (10%),monoOctreotate-bispentylcarboxymethylindo-cyanine dye (Cytate 3, 60%)and bis Octreotate-bispentylcarboxymethyl-indocyanine dye (Cytate 4,30%).

EXAMPLE 11 Formulation of Peptide-Dye Conjugates in Dimethyl Sulfoxide(DMSO)

The dye-peptide conjugates are sparingly soluble in water and requirethe addition of solubilizing agents or co-solvents. Addition of 1-20%aqueous ethanol to the conjugates partially quenched the fluorescenceintensity in vitro and the fluorescence was completely quenched in vivo(the conjugate was not detected by the charge coupled device (CCD)camera). Addition of 1-50% of DMSO either re-established or increasedthe fluorescence intensity of the conjugates in vitro and in vivo. Thedye fluorescence remained intense for over one week. The DMSOformulations were well tolerated by experimental animals used for thisinvention.

EXAMPLE 12 Imaging of Pancreatic Ductal Adenocarcinoma (DSL 6A) withIndocyanine Green (ICG)

A non-invasive in vivo fluorescence imaging apparatus was employed toassess the efficacy of contrast agents developed for tumor detection inanimal models. A LaserMax Inc. laser diode of nominal wavelength 780 nmand nominal power of 40 mW was used. The detector was a PrincetonInstruments model RTE/CCD-1317-K/2 CCD camera with a Rodenstock 10 mm F2lens (stock #542.032.002.20) attached. An 830 nm interference lens (CVILaser Corp., part # F10-830-4-2) was mounted in front of the CCD inputlens such that only emitted fluorescent light from the contrast agentwas imaged. Typically, an image of the animal was taken pre-injection ofcontrast agent. This image was subsequently subtracted (pixel by pixel)from the post injection images. However, the background subtraction wasnever done once the animal had been removed from the sample area andreturned at a later time for images taken several hours post injection.

DSL 6A tumors were induced in male Lewis rats in the left flank area bythe introduction of material from a solid (donor) implant, and thetumors were palpable in approximately 14 days. The animals wereanesthetized with xylazine:ketamine:acepromazine, 1.5:1.5:0.5 at 0.8mL/kg via intramuscular injection. The area of the tumor (left flank)was shaved to expose the tumor and the surrounding surface area. A 21gauge butterfly equipped with a stopcock and two syringes containingheparinized saline was placed into the later tail vein of the rat.Patency of the vein was checked prior to administration of the ICG viathe butterfly apparatus. Each animal received 500 μL of a 0.42 mg/mLsolution of ICG in water.

FIGS. 7A-B are tumor images at two minutes (FIG. 7A) and 30 minutes(FIG. 7B) post bolus injection of a 0.5 mL aqueous solution of ICG (5.4μm). Tetracarboxylic acid cyanine dyes were synthesized as shown in FIG.2, with A=CH₂ or CH₂OCH₂; R₁=R₂=H (Formula 1) or R₁, R₂=fused phenyl(Formula 2).

The Figures are false color images of fluorescent intensity measured atthe indicated times, with images constrained to the tumor and a smallsurrounding area. As is shown, the dye intensity in the tumor isconsiderably diminished 30 minutes post-ICG injection.

EXAMPLE 13 Imaging of Prostatic Carcinoma (R3327-H) with IndocyanineGreen (ICG)

The imaging apparatus and the procedure used are described as in Example12. Prostate tumors (Dunning R3327-H) were induced in young maleCopenhagen rats in the left flank area from a solid implant. Thesetumors grow very slowly and palpable masses were present 4-5 months postimplant. FIGS. 7C-D are images of a rat with an induced prostaticcarcinoma tumor (R3327-H) imaged at two minutes (FIG. 7C) and 30 minutes(FIG. 7D) post injection.

The Figures are false color images of fluorescent intensity measured atthe indicated times, with images constrained to the tumor and a smallsurrounding area. As is shown, the dye intensity in the tumor isconsiderably diminished 30 minutes post-ICG injection.

EXAMPLE 14 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withIndocyanine Green (ICG)

The imaging apparatus and the procedure used are described in Example12. Rat pancreatic acinar carcinoma expressing the SST-2 receptor(CA20948) was induced by solid implant technique in the left flank area,and palpable masses were detected nine days post implant. The imagesobtained at 2 and 30 minutes post injection are shown in FIG. 7E-F.FIGS. 7E-F are images of a rat with an induced pancreatic acinarcarcinoma (CA20948) expressing the SST-2 receptor imaged at two minutes(FIG. 7E) and 30 minutes (FIG. 7F) post injection.

The Figures are false color images of fluorescent intensity measured atthe indicated times, with images constrained to the tumor and a smallsurrounding area. As is shown, the dye intensity in the tumor isconsiderably diminished and almost absent 30 minutes post-ICG injection.

EXAMPLE 15 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withCytate 1

The imaging apparatus and the procedure used are described in Example12, except that each animal received 500 μL of a 1.0 mg/mL solution ofCytate 1 solution of 25% dimethylsulfoxide in water.

Rat pancreatic acinar carcinoma expressing the SST-2 receptor (CA20948)were induced by solid implant technique in the left flank area, andpalpable masses were detected 24 days post implant. Images were obtainedat various times post injection. Uptake into the tumor was seen at twominutes but was not maximal until about five minutes.

FIGS. 8A-B show a comparison of the uptake of ICG and Cytate 1 at 45minutes in rats with the CA20948 tumor cell line. By 45 minutes the ICGhas mostly cleared (FIG. 8A) whereas the Cytate 1 is still intense (FIG.8B). This dye fluorescence remained intense in the tumor for severalhours post-injection.

EXAMPLE 16 Imaging of Rat Pancreatic Acinar Carcinoma (CA20948) withCytate 1 Compared with Imaging with Indocyanine Green

Using indocyanine green (ICG), three different tumor lines were imagedoptically using a CCD camera apparatus. Two of the lines, DSL 6/A(pancreatic) and Dunning R3327H (prostate) indicated slow perfusion ofthe agent over time into the tumor and reasonable images were obtainedfor each. The third line, CA20948 (pancreatic), indicated only a slightbut transient perfusion that was absent after only 30 minutes postinjection. This indicated no non-specific localization of ICG into thisline compared to the other two tumor lines, suggesting a differentvascular architecture for this type of tumor (see FIGS. 7A-F). The firsttwo tumor lines (DSL 6/A and R3327H) are not as highly vascularized asCA20948, which is also rich in somatostatin (SST-2) receptors.Consequently, the detection and retention of a dye in this tumor modelis a good index of receptor-mediated specificity.

Octreotate is known to target somatostatin (SST-2) receptors, hence,cyano-Octreotates (Cytate 1 and Cytate 2) were prepared. Cytate 1 wasevaluated in the CA20948 Lewis rat model. Using the CCD cameraapparatus, strong localization of this dye was observed in the tumor at90 minutes post injection (FIG. 9A). At 19 hours post injection theanimal was again imaged (FIG. 9B). Tumor visualization was easilyobserved showing specificity of this agent for the SST-2 receptorspresent in this tumor line. As a control, the organs were imaged again(FIG. 10A) and the image was compared with that of the same tissues inthe uninjected rat (FIG. 10B).

Individual organs were removed and imaged. High uptake of the materialwas observed in the pancreas, adrenals and tumor tissue, while heart,muscle, spleen and liver indicated significantly lower uptake (FIG. 11).These data correlate well with radiolabeled Octreotate in the same modelsystem (M. de Jong, et al. Cancer Res. 1998, 58, 437-441).

EXAMPLE 17 Monitoring of the Blood Clearance Profile of Peptide-DyeConjugates

A laser of appropriate wavelength for excitation of the dye chromophorewas directed into one end of a fiber optic bundle and the other end waspositioned a few millimeters from the ear of a rat. A second fiber opticbundle was also positioned near the same ear to detect the emittedfluorescent light and the other end was directed into the optics andelectronics for data collection. An interference filter (IF) in thecollection optics train was used to select emitted fluorescent light ofthe appropriate wavelength for the dye chromophore.

Sprague-Dawley or Fischer 344 rats were used in these studies. Theanimals were anesthetized with urethane administered via intraperitonealinjection at a dose of 1.35 g/kg body weight. After the animals hadachieved the desired plane of anesthesia, a 21 gauge butterfly with 12″tubing was placed in the lateral tail vein of each animal and flushedwith heparinized saline. The animals were placed onto a heating pad andkept warm throughout the entire study. The lobe of the left ear wasaffixed to a glass microscope slide to reduce movement and vibration.

Incident laser light delivered from the fiber optic was centered on theaffixed ear. Data acquisition was then initiated, and a backgroundreading of fluorescence was obtained prior to administration of the testagent. For Cytates 1 or 2, the peptide-dye conjugate was administered tothe animal through a bolus injection, typically 0.5 to 2.0 mL, in thelateral tail vein. This procedure was repeated with several dye-peptideconjugates in normal and tumor-bearing rats. Representative profiles asa method to monitor blood clearance of the peptide-dye conjugate innormal and tumor-bearing animals are shown in FIGS. 12 to 16. The datawere analyzed using a standard sigma plot software program for aone-compartment model.

In rats treated with Cytates 1 or 2, the fluorescence signal rapidlyincreased to a peak value. The signal then decayed as a function of timeas the conjugate cleared from the bloodstream. FIG. 12 shows theclearance profile of Cytate 1 from the blood of a normal rat monitoredat 830 nm after excitation at 780 nm. FIG. 13 shows the clearanceprofile of Cytate 1 from the blood of a pancreatic tumor(CA20948)-bearing rat also monitored at 830 nm after excitation at 780nm.

FIG. 14 shows the clearance profile of Cytate 2 from the blood of anormal rat, and FIG. 15 shows the clearance profile of Cytate 2 from theblood of a pancreatic tumor (CA20948)-bearing rat, monitored at 830 nmafter excitation at 780 nm.

FIG. 16 shows the clearance profile of Cytate 4 from the blood of anormal rat monitored at 830 nm after excitation at 780 nm.

It should be understood that the embodiments of the present inventionshown and described in the specification are only specific embodimentsof the inventors who are skilled in the art and are not limiting in anyway. Therefore, various changes, modifications or alterations to thoseembodiments may be made or resorted to without departing from the spiritof the invention and the scope of the following claims. The referencescited are expressly incorporated by reference herein in their entirety.

1. A compound of formula

wherein W⁵ and X⁵ are —S—; Y⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm, —(CH₂)_(a)NR³R⁴,and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consistingof —(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—NHCO—Dm, —CH₂(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm, (CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or adouble bond; B₃, C₃, and D₃ are independently selected from the groupconsisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl, NR³, and —C═O;A₃, B₃, C₃, and D₃ may together form a 6- to 12-membered carbocyclicring or a 6- to 12-membered heterocyclic ring optionally containing oneor more oxygen, nitrogen, or sulfur atom; a₅ varies from 0 to 5; R¹ toR⁴, and R⁵⁸ to R⁶⁶ are independently selected from the group consistingof hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl, C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀aminoalkyl, cyano, nitro, halogen, saccharide, peptide,—CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide, protein, cell, antibody, antibodyfragment, saccharide, glycopeptide, peptidomimetic, drug, drug mimic,hormone, metal chelating agent, radioactive or nonradioactive metalcomplex, and echogenic agent; a and c independently vary from 1 to 20; band d independently vary from 1 to
 100. 2. The compound of claim 1wherein W⁵ and X⁵ are —S—; Y⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, andCH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or a double bond; B₃, C₃, andD₃ are independently selected from the group consisting of —O—, —O—,NR³, alkyl, and —CR¹; A₃, B₃, C₃, and D₃ may together form a 6- to10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ringoptionally containing one or more oxygen, nitrogen, or sulfur atom; a₅varies from 0 to 3; R¹ to R⁴ and R⁵⁸ to R⁶⁶ are independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acidunits, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH andCH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide containing 2 to 30 amino acidunits, antibody, mono- or oligosaccharide, glycopeptide, metal chelatingagent, radioactive or nonradioactive metal complex, and echogenic agent;a and c independently vary from 1 to 10; b and d independently vary from1 to
 30. 3. The compound of claim 2 wherein each of W⁵ and X⁵ is —S—; Y⁵is —(CH₂)₂—CONH—Bm; Z⁵ is —(CH₂)₂—CONH—Dm; A₃ is a single bond; A₃, B₃,C₃, and D₃ together form a 6-membered carbocyclic ring; a₅ is 1; R⁵⁸ isgalactose; each R⁵⁹ to R⁶⁶ is hydrogen; Bm is Octreotate; Dm is bombesin(7-14).
 4. A method for performing a diagnostic or therapeutic procedurecomprising administering to an individual an effective amount of thecompound of formula

wherein W⁵ and X⁵ are —S—; Y⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected fromthe group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm,—(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or adouble bond; B₃, C₃, and D₃ are independently selected from the groupconsisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl, NR³, and —C═O;A₃, B₃, C₃, and D₃ may together form a 6- to 12-membered carbocyclicring or a 6- to 12-membered heterocyclic ring optionally containing oneor more oxygen, nitrogen, or sulfur atom; a₅ varies from 0 to 5; R¹ toR⁴, and R⁵⁸ to R⁶⁶ are independently selected from the group consistingof hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl, C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀aminoalkyl, cyano, nitro, halogen, saccharide, peptide,—CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide, protein, cell, antibody, antibodyfragment, saccharide, glycopeptide, peptidomimetic, drug, drug mimic,hormone, metal chelating agent, radioactive or nonradioactive metalcomplex, and echogenic agent; a and c independently vary from 1 to 20; band d independently vary from 1 to 100, and a pharmaceuticallyacceptable carrier or excipient to form a composition, activating thecompound using light, and performing the diagnostic or therapeuticprocedure.
 5. The method of claim 4 comprising administering to anindividual an effective amount of the compound wherein W⁵ and X⁵ are—S—; Y⁵ is selected from the group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or a double bond; B₃, C₃, andD₃ are independently selected from the group consisting of —O—, —S—,NR³, alkyl, —CR¹R², and —CR¹; A₃, B₃, C₃, and D₃ may together form a 6-to 10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ringoptionally containing one or more oxygen, nitrogen, or sulfur atom; a₅varies from 0 to 3; R¹ to R⁴, and R⁵⁸ to R⁶⁶ are independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acidunits, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide containing 2 to 30 amino acidunits, antibody, mono- or oligosaccharide, glycopeptide, metal chelatingagent, radioactive or nonradioactive metal complex, and echogenic agent;a and c independently vary from 1 to 10; b and d independently vary from1 to
 30. 6. The method of claim 5 comprising administering to anindividual an effective amount of the compound wherein each W⁵ and X⁵ is—S—; Y⁵ is —(CH₂)₂—CONH—Bm; Z⁵ is —(CH₂)₂—CONH—Dm; A₃ is a single bond;A₃, B₃, C₃, and D₃ together form a 6-membered carbocyclic ring; a₅ is 1;R⁵⁸ is galactose; each R⁵⁹ to R⁶⁶ is hydrogen; Bm is Octreotate; Dm isbombesin (7-14).
 7. The method of claim 4 wherein said procedure useslight of wavelength in the region of 350-1300 nm.
 8. The method of claim4 wherein the diagnostic procedure is optical tomography.
 9. The methodof claim 4 wherein the diagnostic procedure is fluorescence endoscopy.10. The method of claim 4 further comprising monitoring a bloodclearance profile of said compound by fluorescence, absorbance or lightscattering wherein light of wavelength in the region of 350-1300 nm isused.
 11. The method of claim 4 wherein said procedure further comprisesa step of imaging and therapy wherein said imaging and therapy isselected from the group consisting of absorption, light scattering,photoacoustic and sonofluoresence technique.
 12. The method of claim 4wherein said procedure is for diagnosing atherosclerotic plaques andblood clots.
 13. The method of claim 4 wherein said procedure comprisesadministering localized therapy.
 14. The method of claim 4 wherein saidtherapeutic procedure comprises photodynamic therapy.
 15. The method ofclaim 4 wherein said therapeutic procedure comprises laser assistedguided surgery for the detection of micrometastases.
 16. The method ofclaim 4 further comprising adding a biocompatible organic solvent to theat a concentration of one to fifty percent to the composition to inhibitin vivo or in vitro fluorescence quenching.
 17. The method of claim 16wherein said compound is dissolved in a medium comprising one to fiftypercent dimethyl sulfoxide.
 18. A composition comprising a cyanine dyebioconjugate of formula

wherein W⁵ and X⁵ are —S—; Y⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Bm,(C₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Bm, —(CH₂)_(a)NR³R⁴,and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consistingof —(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—NHCO—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—(CH₂)_(a)—N(R³)—(CH₂)_(b)—CONH—Dm, —(CH₂)_(a)—N(R³)—(CH₂)_(c)—NHCO—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm,—(CH₂)_(a)—N(R³)—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—N(R³)—CH₂—(CH₂OCH₂)_(d)—NHCO—Dm,—(CH₂)_(a)—NR³R⁴, and —CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or adouble bond; B₃, C₃, and D₃ are independently selected from the groupconsisting of —O—, —S—, —Se—, —P—, —CR¹R², —CR¹, alkyl, NR³, and —C═O;A₃, B₃, C₃, and D₃ may together form a 6- to 12-membered carbocyclicring or a 6- to 12-membered heterocyclic ring optionally containing oneor more oxygen, nitrogen, or sulfur atom; a₅ varies from 0 to 5; R¹ toR⁴ and R⁵⁸ to R⁶⁶ are independently selected from the group consistingof hydrogen, C₁-C₁₀ alkyl, C₅-C₂₀ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀polyalkoxyalkyl, C₁-C₂₀ polyhydroxyalkyl, C₅-C₂₀ polyhydroxyaryl, C₁-C₁₀aminoalkyl, cyano, nitro, halogen, saccharide, peptide,—CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide, protein, cell, antibody, antibodyfragment, saccharide, glycopeptide, peptidomimetic, drug, drug mimic,hormone, metal chelating agent, radioactive or nonradioactive metalcomplex, and echogenic agent; a and c independently vary from 1 to 20; band d independently vary from 1 to 100, and a pharmaceuticallyacceptable carrier or excipient.
 19. The composition of claim 18 whereinW⁵ and X⁵ are —S—, and —Se; Y⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; Z⁵ is selected from the group consisting of—(CH₂)_(a)—CONH—Dm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴; A₃ is a single or a double bond; B₃, C₃, andD₃ are independently selected from the group consisting of —O—, —S—,NR³, alkyl, and —CR¹; A₃, B₃, C₃, and D₃ may together form a 6- to10-membered carbocyclic ring or a 6- to 10-membered heterocyclic ringoptionally containing one or more oxygen, nitrogen, or sulfur atom; a₅varies from 0 to 3; R¹ to R⁴, and R⁵⁸ to R⁶⁶ are independently selectedfrom the group consisting of hydrogen, C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀alkoxyl, C₁-C₁₀ polyhydroxyalkyl, C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀aminoalkyl, mono- or oligosaccharide, peptide with 2 to 30 amino acidunits, —CH₂(CH₂OCH₂)_(b)—CH₂—OH, —(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH and—CH₂—(CH₂OCH₂)_(b)—CO₂H; Bm and Dm are independently selected from thegroup consisting of bioactive peptide containing 2 to 30 amino acidunits, antibody, mono- or oligosaccharide, glycopeptide, metal chelatingagent, radioactive or nonradioactive metal complex, and echogenic agent;a and c independently vary from 1 to 10; b and d independently vary from1 to
 30. 20. The composition of claim 19 wherein each of W⁵ and X⁵ is—S—; Y⁵ is —(CH₂)₂—CONH—Bm; Z⁵ is —(CH₂)₂—CONH—Dm; A₃ is a single bond;A₃, B₃, C₃, and D₃ together form a 6-membered carbocyclic ring; a₅ is 1;R⁵⁸ is galactose; each R⁵⁹ to R⁶⁶ is hydrogen; Bm is Octreotate; Dm isbombesin (7-14).
 21. The compound of claim 1 wherein Y⁵ selected fromthe group consisting of —(CH₂)_(a)—CONH—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm, —(CH₂)_(a)—NHCO—Bm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴.
 22. The compound of claim 1 wherein Z⁵ isselected from the group consisting of —(CH₂)_(a)—CONH—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Dm, —(CH₂)_(a)—NHCO—Dm,—CH₂—(CH₂OCH₂)_(b)—CH₂—NHCO—Dm, —(CH₂)_(a)—NR³R⁴, and—CH₂(CH₂OCH₂)_(b)—CH₂NR³R⁴.
 23. The compound of claim 1 wherein B₃, C₃,and D₃ are independently selected from the group consisting of —O—, —S—,NR³, alkyl, —CR¹R², and —CR¹.
 24. The compound of claim 1 wherein a₅varies from 0 to
 3. 25. The compound of claim 1 wherein R¹ to R⁴ and R⁵⁸to R⁶⁶ are independently selected from the group consisting of hydrogen,C₁-C₁₀ alkyl, C₅-C₁₂ aryl, C₁-C₁₀ alkoxyl, C₁-C₁₀ polyhydroxyalkyl,C₅-C₁₂ polyhydroxyaryl, C₁-C₁₀ aminoalkyl, mono- or oligosaccharide,peptide with 2 to 30 amino acid units, —CH₂(CH₂OCH₂)_(b)—CH₂—OH,—(CH₂)_(a)—CO₂H, —(CH₂)_(a)—CONH—Bm, —CH₂—(CH₂OCH₂)_(b)—CH₂—CONH—Bm,—(CH₂)_(a)—NHCO—Bm, —CH₂(CH₂OCH₂)_(b)—CH₂—NHCO—Bm, —(CH₂)_(a)—OH, and—CH₂—(CH₂OCH₂)_(b)—CO₂H.
 26. The compound of claim 1 wherein Bm and Dmare independently selected from the group consisting of bioactivepeptide containing 2 to 30 amino acid units, antibody, mono- oroligosaccharide, glycopeptide, metal chelating agent, radioactive ornonradioactive metal complex, and echogenic agent.
 27. The compound ofclaim 1 wherein a and c independently vary from 1 to
 10. 28. Thecompound of claim 1 wherein b and d independently vary from 1 to 30.